The inhibition of prostate-specific membrane antigen (PSMA) by tight-binding small-molecule inhibitors has not been fully exploited for cell-capture and detection strategies. Our long-term goal is to develop a novel high-throughput technology to capture, image, and quantify metastatic cells that capitalizes on the potency and specific affinity of tight-binding inhibitors for cell-surface hydrolytic enzymes. The overall objective of this R21 application is to prove the concept that high-affinity irreversible inhibitors of PMSA can be employed to capture prostate cancer cells for detection and quantification. Our central hypothesis for the proposed work is that irreversible small-molecule inhibitors of PSMA tethered to a solid support through a cleavable linker can selectively capture prostate cancer cells that express PSMA for detection and quantification. We plan to test our central hypothesis and accomplish our overall objective of this application by pursuing the following specific aims: (1) develop a cell-capture platform for metastatic prostate cancer based on the structural framework of high-affinity inhibitors of PSMA and (2) develop a nanoparticle fluorescent probe to selectively label prostate cancer cells based on the core structure of high-affinity inhibitors of PSMA. The rationale for undertaking the proposed research is that, once we demonstrate that high-affinity small-molecule inhibitors of PSMA tethered to a solid support can selectively capture PSMA-expressing cells, it will serve as a proof-of-concept for the development of novel diagnostic biosensing technology for metastatic prostate cancer in a subsequent R01 proposal. The expected outcomes of this work will be the development of catch and release platforms based on immobilized inhibitors of PSMA as affinity elements will be developed to capture metastatic cancer cells from blood. Secondly, novel nanoparticle agents for detecting and quantifying selectively captured prostate cancer cells will be developed for fluorescence microscopy and flow cytometry applications. The expected positive impact of these results is that it will ultimately assist clinicians in staging prostate cancer, developing personalized therapy modalities, and monitoring treatment. These accomplishments are important, because this work allow for the development of clinically relevant cell-selecting platforms for the detection and sequestering of metastatic prostate cancer cells.